The purpose of this work is to utilize in vitro models of C. trachomatis persistent infection to (i) understand the molecular basis of chlamydial immune evasion that leads to persistence and chronic disease and, (ii) understand the differences in antibiotic efficacy in eradicating in vitro persistent infection by first-choice antibiotics, and (iii) define new antigens recognized by protective cytotoxic T cells that represent novel vaccine targets. We have performed functional genomic studies on all 15 chlamydial reference strains and over 500 oculogenital clinical isolates focusing on the genes that reside within the pathogen's plasticity zone (PZ); a small polymorphic region located at the replication termination region of the genome. The PZ contains the cytotoxin gene, the phospholipase D gene family, and the tryptophan operon (trpRBA). The trpRBA encodes the Trp repressor and TrpB and TrpA polypeptides that form the heterodimeric channeling tryptophan synthase (TS) enzyme. We found that C. trachomatis strains differing in infection and disease organotropism correlated with either missense or nonsense inactivating mutations in the pathogen's TrpA polypeptide. These mutations resulted in a functional full length TS for all genital isolates and truncated non-functional TS in all ocular strains. This functional genomic analysis has established the unique paradigm between ocular and genital tissue tropic strains based solely on TS functionality. This localized and highly specific genetic diversity is the primary difference in the genomic organization among these otherwise genetically very similar strains strongly implicating the TS a major virulence factor for genital tropic isolates. Moreover, functional TS activity was directly correlated to IFN-gamma resistance in human epithelial cells through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain functional TS capable of using indole for tryptophan biosynthesis. Interestingly, Indole is not made by mammalian cells or chlamydiae raising the possibility that indole is acquired from co-infecting indole producing microflora of the female urogenital tract. Utilizing indole to synthesize tryptophan would allow chlamydiae to escape IFN-gamma mediated immune eradication through the IDO tryptophan deprivation pathway, thereby establishing a mechanism of persistent infection and sustained transmission in the female host. This is the first example of such a unique host-parasite-microflora relationship in the pathogenesis of bacterial disease. Future investigations will focus on the development of a humanized murine animal model to experimentally define this unique host-pathogen-microbial interaction. We are also using the in vitro model of IFN-gamma mediated persistent infection as a novel approach to ascertain differences in normal and persistent growth phenotypes to eradication by first-choice anti-chlamydial antibodies, and as part of a novel antigen discovery approach to identify new chlamydial antigen(s) recognized by catatonic T cells. We have found that azithromycin that the minimal bactericidal concentration (MBC) of azithromycin is 10 fold greater than doxycylcine for eradicating persistent chlamydial infection. Conversely, the MBC doxycycline was 10 greater than azithromycin in eradicating normal chlamydial growth. These findings suggest that a combination of these anti-infectives might be more efficacious in managing chlamydial infections, such as trachoma and STD, than the current regime of single antibiotic treatment. Cytotoxic CD8 and CD4 T cells are important effector lymphocyte subsets in protective immunity yet the antigens recognized by these cytotoxic T cells remain largely uncharacterized. Interestingly, HLA class II is induced by IFN-gamma in chlamydial infected human cervical epithelial cells and chlamydial antigens are processed and presented by in the context HLA class II in persistently infected epithelial cells. Thus this unique target of infection represents a novel target for cytotoxic T cells and the HLA antigen(s) presented by these cells are potentially new and important chlamydial vaccine targets. We are purifying HLA class I and II molecules from IFN-gamma treated chlamydial infected cervical epithelial cells, eluting the HLA bound peptides, and identifying the peptides by high resolution mass spectrometry. Peptide sequences are blasted against the chlamydial genome databases to identify the genes from which the peptides are derived. To date, we have identified 30 novel HLA class I antigens, and two novel HLA class II peptides using this approach. Future work will focus on describing the antigenicity of the proteins using isolated T cells obtained from naturally infected humans, generating specific T cells clones, and identifying clones that exhibit chlamydial specific cytotoxic function. The genes encoding these proteins will be cloned and expressed in attenuated vaccinia and influenza viruses and tested as novel genital or intranasal administered vaccines in a pre-clinical model of chlamydial ocular and genital tract-infection.